This invention relates to the field of molecular biology. In particular, it relates to processes involving the propagation and analysis of microorganisms including recombinant microorganisms.
Rehydratable dry film plating media have been described (e.g., U.S. Pat. No. 4,565,783) for the detection and enumeration of microorganisms in food or other samples. For example, dry film plating media technology has been used for testing for a particular antigenic strain of pathogenic E. coli. See, for example, U.S. Pat. No. 5,137,812. Typical devices that use dry film media technology contain a cold-water-soluble gelling system, which allows for addition of a 1 ml aqueous sample. Such devices promote the growth of target organisms within the device under suitable incubation conditions, and also provide a detection system within the device to allow for visualization and enumeration of the colonies growing in the device. These devices have particular utility within the food processing industry, where the detection and enumeration of specific target organisms or indicator organisms serves as an index of food quality and/or safety.
In contrast to microbiological testing, where organisms are seldom stored for future use, it is common among molecular biologists to store recombinant microorganisms in broth cultures or on semisolid media for future use. Additional steps are needed to transfer the recombinant microorganisms from the primary plating medium to the storage medium and, frequently, an additional growth period (16-24 hours) is needed to incubate the storage medium before it is placed at the storage temperature.
A disadvantage of semisolid medium (agar) when used for culture storage is the tendency for the moisture to evaporate from the gel. In the case of unsealed petri dishes, this results in the dehydration of the agar and the death of the cultures. In the case of sealed petri dishes, this results in the condensation of moisture onto the plastic or glass dishes, which may result in moisture spreading across the surface of the agar and cross-contamination of colonies on the plate. Furthermore, it is not possible to freeze cultures on agar plates because the agar tends to develop ice crystals on the surface and/or split when frozen. Both of these features increase the probability of colony cross-contamination, which is undesirable when trying to maintain pure clones of recombinant organisms. In addition, a primary objective of plating recombinant organisms is to perform genetic analyses, which typically results in the destruction of the cells. Thus, it is usually necessary to pick each colony and xe2x80x9creplicatexe2x80x9d it in broth or semisolid media before using the remnants of the colony for the genetic analysis.
The invention described herein is based on novel formulations of dry film plating media that provide unique properties to a device for the propagation or storage of microorganisms. Upon opening the device containing such a formulation, a portion of a colony growing on the plate transfers to both films, providing two replicates of each clone. In addition, colonies on the plate are larger than those growing on commercially available Petrifilm(trademark) plates. These properties offer several advantages to molecular biologists and others who need to analyze or subculture bacterial colonies grown on semisolid media.
In one aspect, the invention features a device for the propagation or storage of microorganisms. The device includes first and second layers, wherein the first layer includes a gelling agent and microbial growth medium and the second layer includes a gelling agent, wherein the device further includes an indicator and a corresponding inducer, and wherein the first and second layers are separable from each other. The first or second layers can be rehydratable. At least 80% (e.g., 85% or 95%) of visible microorganism colonies can partition to form replicates on the first and the second layers upon separation of the layers. The replicates on the first or second layer are detectable by magnified or unmagnified visual inspection, or by genetic analysis. Detection by genetic analysis can include hybridization, polymerase chain reaction, plasmid restriction analysis, or expression screening techniques.
Replicates on the first or second layer are viable. The gelling agent of the first or second layers can be guar gum, xanthan gum, locust bean gum, polyvinyl alcohol, carboxymethylcellulose, alginate, polyvinylpyrolidone, gellan or low monomer content polyacrylic acid. Guar gum is particularly useful.
The microbial growth medium can include a detergent such as an ionic detergent, e.g., deoxycholate, bile salts or lauryl sulfate or a salt. The first layer further can include a selectable agent such as an antibiotic or an amino acid deficiency. Indicators can be precipitable or chromogenic (e.g., 5-bromo-4-chloro-3-indoxyl-xcex2-D-glucuronic acid, L-Alanine-5-bromo-4-chloro-3-indoxyl ester (trifluoroacetate salt), 5-bromo-4-chloro-3-indoxyl-1-acetate, 5-bromo-4-chloro-3-indoxyl-3-acetate, 5-bromo-4-chloro-3-indoxyl-N-acetyl-xcex2-D-galactosaminide, 5-bromo-4-chloro-3-indoxyl-N-acetyl-xcex2-D-glucosaminide, 5-bromo-4-chloro-3-indoxyl butyrate, 5-bromo-4-chloro-3-indoxyl caprylate, 5-bromo-4-chloro-3-indoxyl-xcex2-D-cellobioside, 5-bromo-4-chloro-3-indoxyl-xcex1-L-fucopyranoside, 5-bromo-4-chloro-3-indoxyl-xcex2-D-fucopyranoside, 5-bromo-4-chloro-3-indoxyl-xcex2-L-fucopyranoside, 5-bromo-4-chloro-3-indoxyl-xcex1-D-galactopyranoside, 5-bromo-4-chloro-3-indoxyl-xcex2-D-galactopyranoside, 5-bromo-4-chloro-3-indoxyl-xcex1-D-glucopyranoside, 5-bromo-4-chloro-3-indoxyl-xcex2-D-glucopyranoside, 5-bromo-4-chloro-3-indoxyl-xcex2-D-glucuronic acid (cyclohexylammonium salt), 5-bromo-4-chloro-3-indoxyl-xcex2-D-glucuronicacid (sodium salt), 5-bromo-4-chloro-3-indoxyl myo-inositol-1-phosphate (ammonium salt), 5-bromo-4-chloro-3-indoxyl-xcex1-D-maltotriose, 5-bromo-4-chloro-3-indoxyl myristate, 5-bromo-4-chloro-3-indoxyl-xcex1-D-mannopyranoside, 5-bromo-4-chloro-3-indoxyl-nonano ate, 5-bromo-4-chloro-3-indoxyl oleate, 5-bromo-4-chloro-3-indoxyl palmitate, 5-bromo-4-chloro-3-indoxyl phosphate (di{2-amino-2-methyl-1,3-propanediol}salt), 5-bromo-4-chloro-3-indoxyl phosphate (dilithium salt hydrate), 5-bromo-4-chloro-3-indoxyl phosphate (dipotassium salt), 5-bromo-4-chloro-3-indoxyl phosphate (disodium salt sesquihydrate), 5-bromo-4-chloro-3-indoxyl phosphate (potassium salt), 5-bromo-4-chloro-3-indoxyl phosphate (p-toluidine salt), 5-bromo-4-chloro-3-indoxyl sulfate (potassium salt), 5-bromo-4-chloro-3-indoxyl sulfate (p-toluidine salt), 5-bromo-4-chloro-3-indoxyl thymidine-3xe2x80x2-phosphate (cyclohexylammonium salt) or 5-bromo-4-chloro-3-indoxyl-xcex2-D-xylopyranoside). The inducer can be 1-O-methylglucuronic acid, isopropyl-xcex2-D-thioglucuronic acid, isopropyl-xcex2-D-thiogalactopyranoside, or 1-O-methyl-xcex2-D-glucopyranoside. The indicator and corresponding inducer can be in the same layer.
The first or second layers further can include an adhesive. The first and second layers can include water impermeable substrates such as plastic, glass, or coated paper. For example, the water impermeable substrate can be polystyrene, polyethylene, polypropylene (e.g., biaxially-oriented polypropylene), or polyester. The substrates can include a contiguous piece of material having a fold whereby the first and second layers are substantially opposed to each other. The substrates can be removably or permanently attached to each other such as by a hinge, a clasp, glue, staples, or a clamp.
The microorganisms can be bacteria, fungi, yeast, phage, or mycoplasma. The bacteria can be aerobic, anaerobic or microaerophilic, and gram negative. For example, the bacteria can be E. coli, Staphylococcus, or Pseudomonas.
The invention also features a method for simultaneously propagating and obtaining replicas of a microorganism colony forming unit. The method includes applying an inoculum that includes a microorganism colony forming unit to a device to form an inoculated device, wherein the device includes a first layer that includes a gelling agent and microbial growth medium and a second layer that includes a gelling agent, wherein the first and second layers are separable from each other; contacting the first and second layers of the inoculated device to form a gel; incubating the inoculated device for a time sufficient for at least one cell division; separating the first and second layers to provide replicas of the microorganism colony forming unit; and confirming separation of the microorganism colony forming unit. The device can include an indicator and a corresponding inducer. The method further can include performing a molecular biology manipulation on at least one of the replicates.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.